ABSTRACT Mammalian platelets are small anucleate blood cells specialized to continuously monitor and preserve the integrity of the cardiovascular system (hemostasis). They are produced by megakaryocytes (MKs) in the bone marrow and released into blood, where they circulate for ten days in humans and five days in mice until they get cleared by phagocytes. Platelet homeostasis, i.e. the establishment of a defined peripheral platelet count, requires tight regulation of both platelet production and clearance. To fulfill their hemostatic function, platelets depend on a very sensitive signaling machinery that facilitates platelet adhesion under shear stress. This high sensitivity, however, poses a risk for unwanted platelet activation that can lead to platelet clearance and/or thrombosis. The overarching goal of our work is to achieve a better understanding of the molecular mechanisms regulating MK development and platelet reactivity, with a specific focus on the role of small GTPases in these processes. This R35 OIA application is an extension to three funded NHLBI R01 grants: Small GTPases in Megakaryocyte Biology; Rap1 Signaling in Platelet Homeostasis and Vascular Hemostasis; Spatial Regulation of Platelet Activation by Podoplanin-Clec2 Signaling. Our MK studies utilize unique biosensors to establish a molecular signature of small GTPase activity (both Rho and Rap GTPases) during the final stages of development, including the transition from proliferation to proplatelet formation. Once established, we will establish proof-of-principle that precisely targeted perturbation of GTPase activity by optogenetic tools is a viable strategy to optimize in vitro platelet production, a hot topic in Transfusion Medicine. Our platelet work focuses more specifically on the role of Rap GTPases as master regulators of cellular activation and hemostatic plug formation. We have utilized unique mouse models to establish the importance and the key regulators of Rap1 signaling during platelet activation. Furthermore, we have shown that Rap1 activity has to be tightly balanced both in quiescent, circulating and in hemostatically active platelets, and that disturbance of this pathway leads to bleeding or thrombocytopenia/ thrombosis. In ongoing and future work, we will expand on our cell biological and biochemical/-physical studies to provide a comprehensive understanding of how Rap signaling controls platelet function, how it is regulated, and if/ how it contributes to other patho-physiological processes such as vascular integrity in development/ inflammation and venous thrombosis. We will use our unique biochemical assays to screen for inhibitors of Rap signaling. Our clinical studies will investigate if Rap1 signaling is altered in various pathologies, and whether there is interindividual variability in this pathway in healthy and diseased subjects? Together, these studies are expected to lead to novel strategies for the diagnosis and management of some inherited and acquired thrombocytopenias and bleeding disorders, and to a more personalized approach to anti-platelet therapy.